Differences in the mannose oligomer specificities of the closely related lectins from Galanthus nivalis and Zea maysstrongly determine their eventual anti-HIV activity
© Hoorelbeke et al; licensee BioMed Central Ltd. 2011
Received: 22 September 2010
Accepted: 11 February 2011
Published: 11 February 2011
In a recent report, the carbohydrate-binding specificities of the plant lectins Galanthus nivalis (GNA) and the closely related lectin from Zea mays (GNAmaize) were determined by glycan array analysis and indicated that GNAmaize recognizes complex-type N-glycans whereas GNA has specificity towards high-mannose-type glycans. Both lectins are tetrameric proteins sharing 64% sequence similarity.
GNAmaize appeared to be ~20- to 100-fold less inhibitory than GNA against HIV infection, syncytia formation between persistently HIV-1-infected HuT-78 cells and uninfected CD4+ T-lymphocyte SupT1 cells, HIV-1 capture by DC-SIGN and subsequent transmission of DC-SIGN-captured virions to uninfected CD4+ T-lymphocyte cells. In contrast to GNA, which preferentially selects for virus strains with deleted high-mannose-type glycans on gp120, prolonged exposure of HIV-1 to dose-escalating concentrations of GNAmaize selected for mutant virus strains in which one complex-type glycan of gp120 was deleted. Surface Plasmon Resonance (SPR) analysis revealed that GNA and GNAmaize interact with HIV IIIB gp120 with affinity constants (KD) of 0.33 nM and 34 nM, respectively. Whereas immobilized GNA specifically binds mannose oligomers, GNAmaize selectively binds complex-type GlcNAcβ1,2Man oligomers. Also, epitope mapping experiments revealed that GNA and the mannose-specific mAb 2G12 can independently bind from GNAmaize to gp120, whereas GNAmaize cannot efficiently bind to gp120 that contained prebound PHA-E (GlcNAcβ1,2man specific) or SNA (NeuAcα2,6X specific).
The markedly reduced anti-HIV activity of GNAmaize compared to GNA can be explained by the profound shift in glycan recognition and the disappearance of carbohydrate-binding sites in GNAmaize that have high affinity for mannose oligomers. These findings underscore the need for mannose oligomer recognition of therapeutics to be endowed with anti-HIV activity and that mannose, but not complex-type glycan binding of chemotherapeutics to gp120, may result in a pronounced neutralizing activity against the virus.
Lectins represent a heterogeneous group of carbohydrate-binding proteins that are present in different species (e.g. prokaryotes, plants, invertebrates and vertebrates) and vary in size, structure and ability (affinity for different glycan determinants) to bind carbohydrates. Plant lectins represent a large group of proteins classified into twelve families, each typified by a particular carbohydrate binding motif . At present, most studies have dealt with plant lectins classified as legume lectins, chitin-binding lectins, type 2 ribosome inactivating proteins and monocot mannose-binding lectins (MMBLs). After the identification of the first reported MMBL from snowdrop bulbs, namely Galanthus nivalis agglutinin (GNA) , lectins were isolated and characterized from other closely related plant species. Similar lectins were also identified outside plants, for example in the fish Fugu rubripes  and in several Pseudomonas spp. [4, 5]. GNA is the prototype of a family of lectins that resemble each other with respect to their amino acid sequences, three-dimensional structures, and sugar-binding specificities. The lectin subunits of this class contain similar structural features, containing a β-barrel composed of 3 antiparallel four-stranded β sheets .
Members of the GNA-related lectins have been investigated for their antiviral activity (in particular HIV). Indeed, the plant lectins Galanthus nivalis agglutinin (GNA) and Hippeastrum hybrid agglutinin (HHA) have been described to inhibit viral entry [7, 8], presumably by their interaction with the glycans on HIV gp120. It has been reported that these carbohydrate binding agents (CBAs) block virus entry by inhibiting the fusion of cell-free HIV particles with their target cells. Also, they prevent the capture of virions by the DC-SIGN-receptor present on dendritic cells of the innate immune system and efficiently inhibit the subsequent transmission of the virus to CD4+ T-cells. Besides blocking HIV entry, CBAs have also the ability to select for virus strains in which one or more glycans on gp120 are deleted. This mechanism of drug escape results in the exposure of previously hidden immunogenic epitopes on the virus envelope glycoproteins .
Until recently, most plant lectin research was limited to vacuolar plant lectins which have the advantage of being present at relatively high quantities in seeds. Nowadays, nucleocytoplasmic plant lectins can also be efficiently isolated, even though they occur at low concentrations in the plant tissues. One example of a nucleocytoplasmic plant lectin is the maize homolog of the vacuolar GNA . This GNA-like lectin from Zea mays (GNAmaize) of which the gene was cloned and expressed in Pichia pastoris by Fouquaert and co-workers  shows 64% sequence similarity with GNA from snowdrop.
All the reported GNA-related lectins including GNAmaize have homologous sequences and structural similarities. Despite this similarity at the protein level, this class of lectins may display important differences in the post-translational processing of the precursors . Many GNA-related lectins are indeed synthesized as prepro-proteins and then converted in the mature polypeptide by the co-translational cleavage of a signal peptide and the post-translational removal of a C-terminal peptide . However, more recently it was shown that some GNA-related lectins are synthesized without a signal peptide and as a consequence are located in the nucleocytoplasmic compartment of the plant cell. This processing results in a different subcellular localization of the lectin. The GNA homolog from maize (GNAmaize) is processed in such a way and is, therefore, in contrast to the vacuolar GNA, located in the cytoplasm [10, 11].
Native GNA is a tetrameric protein of 50 kDa with three carbohydrate-binding motifs in each monomer and was originally isolated from snowdrop bulbs . GNA was originally described as a lectin with a specificity towards Manα1,3Man-containing oligosaccharides . The molecular mass of the native recombinant GNAmaize is 60 kDa and the lectin exists also as a tetramer with 3 carbohydrate-binding sites per monomer . However, it was reported before that gene divergence may have a serious impact on the carbohydrate-binding potential of lectins . Sequence alignments revealed that only the third carbohydrate-binding site (CBS) is similar between the GNAmaize and the GNA lectin, whereas the first and second CBS differ with only 2 and 1 amino acid changes, respectively . However, glycan microarray analysis revealed striking differences in glycan specificity. GNAmaize interacts preferentially with complex-type glycans, whereas GNA almost exclusively binds to high-mannose-type glycans . Fouquaert and colleagues hypothesized that this difference in glycan-binding properties reflects the ~100-fold decreased anti-HIV-1 activity of GNAmaize when compared to GNA .
To reveal in more detail the correlation between gene divergency of GNA and GNAmaize, as well as the change in carbohydrate-binding specificity and differences in anti-HIV activity, we now report a detailed study of GNAmaize (in comparison with GNA) covering its anti-HIV activity, its kinetic interaction with the HIV-1 envelope glycoprotein gp120, epitope mapping experiments to determine its glycan specificity on gp120 and its antiviral resistance spectrum.
The mannose-specific plant lectin GNA from snowdrop and the cytoplasmatic GNAmaize from maize were derived and purified as described previously [2, 11]. GlcNAcß1,2Man, (α1,3-man)2 and (β1,4-GlcNAc)3 were obtained from Dextra Laboratories (Reading, UK). (α1,2-man)3 was purchased from Carbohydrate Synthesis (Oxford, UK). The anti-gp120 2G12 mAb was obtained from Polymun Scientific GmbH (Vienna, Austria). The lectins Phaseolus vulgaris Erythroagglutinin (PHA-E) and Sambucus nigra agglutinin (SNA) from elderberry were from Vector Laboratories (Peterborough, UK).
Human T-lymphocytic CEM, C8166, HuT-78 and Sup-T1 cells were obtained from the American Type Culture Collection (Manassas, VA, USA). The Raji/DC-SIGN cells were constructed by Geijtenbeek et al.  and kindly provided by L. Burleigh (Institut Pasteur, Paris, France). Persistently HIV-infected HuT-78/HIV cells were obtained upon cultivation for 3 to 4 weeks of HuT-78 cell cultures exposed to HIV-1(IIIB). All cell lines were cultivated in RPMI-1640 medium (Invitrogen, Merelbeke, Belgium) supplemented with 10% fetal bovine serum (FBS) (BioWittaker Europe, Verviers, Belgium), 2 mM L-glutamine, 75 mM NaHCO3 and 20 μg/ml gentamicin (Invitrogen).
HIV-1(IIIB) and HIV-1(BaL) were a kind gift from R.C. Gallo (Institute of Human Virology, University of Maryland, Baltimore, MD) (at that time at the NIH, Bethesda, MD) and HIV-2(ROD) was provided by L. Montagnier (at that time at the Pasteur Institute, Paris, France). The following clinical isolates were used: UG273 (clade A, R5), DJ259 (clade C, R5) and ID12 (clade A/E, R5).
CEM cells (5 × 105 cells per ml) were suspended in fresh culture medium and infected with HIV-1 and HIV-2 at 100 times the CCID50 (50% cell culture infective doses) per ml of cell suspension, of which 100 μl was mixed with 100 μl of the appropriate dilutions of the test compounds, and further incubated at 37°C. After 4 to 5 days, syncytia formation was recorded microscopically in the cell cultures. The 50% effective concentration (EC50) corresponds to the compound concentration required to prevent syncytium formation by 50% in the virus-infected CEM cell cultures.
Buffy coat preparations from healthy donors were obtained from the Blood Bank in Leuven. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation over Lymphoprep (density = 1.077 g/ml; Nycomed, Oslo, Norway). The PBMC were transferred to RPMI 1640 medium supplemented with 10% fetal calf serum (BioWhittaker Europe) and 2 mM L-glutamine and then stimulated for 3 days with phytohemagglutinin (PHA; Murex Biotech Limited, Dartford, United Kingdom) at 2 μg/ml. HIV-infected or mock-infected PHA-stimulated blasts were cultured in the presence of 10 ng of interleukin-2/ml and various concentrations of GNA and GNAmaize. Supernatant was collected at days 8 to 10, and HIV-1 core antigen in the culture supernatant was analyzed by the p24 core antigen enzyme-linked immunosorbent assay (ELISA; DuPont-Merck Pharmaceutical Co., Wilmington, Del.).
Co-cultivation assay between Sup-T1 and persistently HIV-1-infected HuT-78 cells
Persistently HIV-1(IIIB)-infected HuT-78 cells (designated HuT-78/HIV-1) were washed to remove cell-free virus from the culture medium, and 5 × 104 cells (50 μl) were transferred to 96-well microtiter plates. Next, a similar amount of Sup-T1 cells (50 μl) and appropriate concentrations of test compound (100 μl), were added to each well. After 1 to 2 days of co-culturing at 37°C, the EC50 values were quantified based on the appearance of giant cells by microscopical inspection.
Capture of HIV-1(IIIB) by Raji/DC-SIGN cells and subsequent co-cultivation with C8166 cells
The experiment was performed as described previously . Briefly, B-lymphocyte DC-SIGN-expressing (Raji/DC-SIGN) cells were suspended in cell culture medium at 2 × 106 cells/ml. 100 μl of HIV-1(IIIB) (~250,000 pg p24) were added in the presence of 400 μl of serial dilutions of the test compounds. After 60 minutes of incubation, the cells were carefully washed 3 times to remove unbound virions and resuspended in 1 ml of cell culture medium. The captured HIV-1(IIIB) was quantified by a p24 Ag ELISA. From the Raji/DC-SIGN cell suspension, 200 μl were also added to the wells of a 48-well microtiter plate in the presence of 800 μl uninfected C8166 cells (2.5 × 105 cells/ml). These cocultures were further incubated at 37°C, and syncytia formation was evaluated microscopically after ~ 18 to 42 h, and viral p24 Ag determination in the culture supernatants was performed.
Selection and isolation of GNAmaize-resistant HIV-1 strains
CEM cells were infected with HIV-1(IIIB) and seeded in 48-well plates in the presence of GNAmaize at a concentration equal to one- to two-fold its EC50. Three independent series of subcultivations were performed for GNAmaize. The compound concentration was increased stepwise (~ 1.5-fold) when full cytopathic effect was detected. Subcultivations occurred after every 4 to 5 days by transferring 100 μl cell suspension of the GNAmaize-exposed HIV-infected cells to 900 μl uninfected CEM cell cultures.
Genotyping of the HIV-1 env region
Viral RNA was extracted from virus supernatants using the QIAamp Viral RNA Mini Kit (Westburg, Heusden, the Netherlands). The genotyping of both Env genes, gp120 and gp41, were determined in this assay as described previously .
Surface plasmon resonance (SPR) analysis
Recombinant gp120 proteins from HIV-1(IIIB) (ImmunoDiagnostics Inc., Woburn, MA), one batch produced by CHO cell cultures and another by insect cells (Baculovirus) were covalently immobilized on a CM5 sensor chip in 10 mM sodium acetate, pH 4.0, using standard amine coupling chemistry. The exact chip densities are summarised in the results section. A reference flow cell was used as a control for non-specific binding and refractive index changes. All interaction studies were performed at 25°C on a Biacore T100 instrument (GE Healthcare, Uppsala, Sweden). The plant lectins GNA and GNAmaize were serially diluted in HBS-P (10 mM HEPES, 150 mM NaCl and 0.05% surfactant P20; pH 7.4) supplemented with 0.2 mM Ca2+, covering a wide concentration range by using two-fold dilution steps. Samples (often in duplicate) were injected for 2 minutes at a flow rate of 45 μl/min and the dissociation was followed for 8 minutes. Several buffer blanks were used for double referencing. The CM5 sensor chip surface was regenerated with 1 injection of 50 mM NaOH and with 1 injection of Glycine-HCl pH 1.5 for GNAmaize and GNA, respectively. All studied interactions resulted in specific binding signals. The shape of the association and dissociation phases reveals that the curves are not following 1:1 Langmuir kinetics. The experimental data were fit using the 1:1 binding model (Biacore T100 Evaluation software 2.0.2) to determine the binding kinetics. These affinity and kinetic values are apparent values as the injected concentrations of the evaluated compounds did result in biphasic binding signals.
To generate more information on the glycan specificity of GNAmaize and GNA, three different SPR-based experiments were performed. In the first set-up, the sensor chip was immobilized with GNA and GNAmaize and binding with the (α1,2-man)3, (α1,3-man)2, (β1,4-GlcNAc)3, and GlcNAcß1,2Man analytes was examined as described above. The experimental data were fit using the steady-state affinity model (Biacore T100 Evaluation software 2.0.2) to determine the apparent KD-values. In the second set-up, a competition assay of GNAmaize, GNA and the anti-gp120 2G12 mAb for binding to immobilized HIV-1 gp120 was performed in which one of each of the compounds was administered for 2 minutes to immobilized gp120 and by the end of this time period, the initial compound concentration was sustained but now in the additional presence of one of the two other compounds. In a third set-up, a competition experiment for binding of GNA, GNAmaize and the mAb 2G12 to HIV-1 gp120 was performed with PHA-E (prefers binding to GlcNAcß1,2man- and Galß1,4GlcNAc determinants) and SNA (prefers binding to NeuAcα2,6- and to a lesser degree NeuAcα2,3-X determinants).
Homology modeling of GNAmaize was performed on a Silicon Graphics O2 10000 workstation, using the programs InsightII, Homology and Discover (Accelrys, San Diego CA, USA). The atomic coordinates of GNA complexed to mannose (code 1MSA)  were taken from the RCSB Protein Data Bank  and used to build the three-dimensional model of the GNA-like lectin from maize. The amino acid sequence alignment was performed with CLUSTAL-X  and the Hydrophobic Cluster Analysis (HCA)  plot was generated http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal.py?form=HCA to recognize the structurally conserved regions common to GNA and GNAmaize. Steric conflicts resulting from the replacement or the insertion of some residues in the modeled lectin were corrected during the model building procedure using the rotamer library  and the search algorithm implemented in the Homology program  to maintain proper side-chain orientation. Energy minimization and relaxation of the loop regions were carried out by several cycles of steepest descent using Discover3. After correction of the geometry of the loops using the minimize option of TurboFrodo, a final energy minimization step was performed by 100 cycles of steepest descent using Discover 3, keeping the amino acid residues forming the carbohydrate-binding sites constrained. The program TurboFrodo (Bio-Graphics, Marseille, France) was used to draw the Ramachandran plots  and perform the superimposition of the models. PROCHECK  was used to check the stereochemical quality of the three-dimensional model: 74.8% of the residues were assigned to the most favourable regions of the Ramachandran plot (77.6% for GNA). Cartoons were drawn with Chimera .
Molecular surface and electrostatic potentials were calculated and displayed with GRASP using the parse3 parameters . The solvent probe radius used for molecular surfaces was 1.4 Å and a standard 2.0 Å-Stern layer was used to exclude ions from the molecular surface . The inner and outer dielectric constants applied to the protein and the solvent were fixed at 4.0 and 80.0, respectively, and calculations were performed keeping a salt concentration of 0.145 M. Surface topology of the carbohydrate-binding sites was rendered and analyzed with PyMol (W.L. DeLano, http://pymol.org).
The docking of methyl mannose (MeMan) into the carbohydrate-binding sites of GNAmaize was performed with the program InsightII (Accelrys, San Diego CA, USA). The lowest apparent binding energy (Ebind expressed in kcal.mol-1) compatible with the hydrogen bonds (considering Van de Waals interactions and strong [2.5 Å < dist(D-A) < 3.1 Å and 120° < ang(D-H-A)] and weak [2.5 Å < dist(D-A) < 3.5 Å and 105° < ang(D-H-A) < 120°] hydrogen bonds; with D: donor, A: acceptor and H: hydrogen) found in the GNA/Man complex (RCSB PDB code 1MSA)  was calculated using the forcefield of Discover3 and used to anchor the pyranose ring of the sugars into the binding sites of the lectin. The positions of mannose observed in the GNA/Man complex were used as starting positions to anchor mannose in the carbohydrate-binding sites of GNAmaize. Cartoons showing the docking of MeMan in the mannose-binding sites of the lectins were drawn with Chimera and PyMol.
Antiviral activity of GNA and GNAmaize against HIV-1(IIIB) and HIV-2(ROD) infection
Anti-HIV activity of GNAmaize and GNA in different cell systems
HuT-78/HIV-1 + Sup T1 EC50b(μM)
0.46 ± 0.13
0.007 ± 0.001
0.008 ± 0.001
0.062 ± 0.064
Antiviral activity of GNAmaize and GNA in PBMC against clinical isolates
Clade A, UG273
Clade B, BaL
Clade C, DJ259
Clade A/E, ID12
Activity of CBAs on syncytia formation in co-cultures between HuT-78/HIV-1 and Sup-T1 cells
GNAmaize could not efficiently prevent syncytia formation between persistently HIV-1(IIIB)-infected HuT-78/HIV-1 cells and uninfected CD4+ T-lymphocyte SupT1 cells (EC50 >1.7 μM), whereas GNA was able to prevent syncytia formation in the co-cultures at an EC50 of 0.062 μM (Table 1 and Figure 1, Panel C).
Effect of GNA and GNAmaize on the capture of HIV-1 by Raji/DC-SIGN cells and on subsequent virus transmission to uninfected CD4+T-cells
Inhibitory activity of GNAmaize and GNA on DC-SIGN-mediated capture of HIV-1(IIIB) by DC-SIGN+ cells and subsequent virus transmission to CD4+ T cells
0.90 ± 0.40
0.44 ± 0.09
0.04 ± 0.01
0.006 ± 0.005
Selection of GNAmaize -resistant HIV-1(IIIB) strains and determination of mutations in the gp160 gene of GNAmaize-exposed HIV-1(IIIB) strains
Amino acid mutations that appeared in the envelope of HIV-1(IIIB) strains under sustained GNAmaize or GNA pressure
putative glycosylation motifs in HIV-1(IIIB) gp160
type of N-glycan
Kinetic analysis of the interaction of GNA and GNAmaize with HIV-1 IIIBgp120
Kinetic data for the interaction of GNA and GNAmaize with immobilized HIV-1 IIIB gp120
GNA vs III B gp120 (CHO)
0.33 ± 0.07
(2.81 ± 0.68) E+06
(9.00 ± 1.14) E-04
GNA vs III B gp120 (Baculovirus)
0.17 ± 0.12
(2.75 ± 1.56) E+06
(3.63 ± 0.75) E-04
GNA maize vs III B gp120 (CHO)
34 ± 13
(1.37 ± 0.78) E+05
(5.24 ± 4.50) E-03
GNA maize vs III B gp120 (Baculovirus)
77 ± 17
(2.23 ± 0.74) E+04
(1.64 ± 0.20) E-03
Affinity analysis for the interactions of various oligosaccharides with GNAmaizeand GNA
Affinity data for the interactions of various oligosaccharides with immobilized GNA and GNAmaize
1.5 ± 0.2 mM
4.4 ± 0.9 mM
Competition of GNA, GNAmaizeand mAb 2G12 for binding to HIV-1 gp120
Competition between PHA-E or SNA and GNA, GNAmaizeor mAb 2G12 for binding to HIV-1 gp120
Homology modeling of GNAmaize
Our antiviral data and previous observations  revealed that GNA and GNAmaize both inhibit HIV-1 and HIV-2 infection. However GNAmaize shows a strongly reduced anti-HIV-activity compared to GNA, being ~60- to ~100-fold less potent against HIV-1(IIIB) and HIV-2(ROD) infection. It was 30-fold inferior to inhibit giant cell formation between persistently HIV-1-infected HuT-78 cells and uninfected SupT1 cells, and it was 20- to 70-fold less efficient in inhibiting DC-SIGN-directed HIV-1 capture and subsequent transmission of DC-SIGN-captured HIV-1 particles to uninfected CD4+ T-lymphocytes (Tables 1, 2, 3). The decreased antiviral activity is in agreement with the much lower affinity [~ 100-fold higher apparent affinity constant (KD)] that was recorded for the interaction between GNAmaize and gp120 compared to GNA and gp120. This value points to a ~ 100-fold weaker binding of GNAmaize than GNA to gp120. Thus, despite the high similarities at the sequence and structural level, both plant lectins have a strikingly different potency for their anti-HIV activity and interaction with their antiviral target (HIV gp120). Thus, the weaker contribution to the inhibitory effect against the HIV-1 infection by GNAmaize is closely correlated with its weaker binding to HIV-1 gp120, presumably due to its carbohydrate specificity shift from oligomannose (for GNA) to complex-type glycans. In this respect, it cannot be excluded that the anti-HIV activity of GNAmaize may be due, not only to a binding to complex-type glycans present on HIV-1 gp120 but also to potential binding to complex-type glycans of gangliosides that may be present in the virion envelope.
In the long-term drug selection experiments with GNAmaize, one N-glycan deletion in gp120 (N301) was observed when all virus strains were taken into account (Table 4). The deletion represents a complex-type glycan deletion . This N-linked sugar chain is the only one present in the V3-loop of the HIV-1 envelope. This complex-type N-glycan is conserved in most HIV-1 strains. The N301 glycan is in close proximity to important protein domains, in contrast to the complex glycans at V1/V2 or V4 of gp120. The V3 loop has been implicated in the binding of gp120 with CD4 and the chemokine secondary receptors . It also plays a role in eliciting neutralizing anti-HIV antibodies [32, 33]. Interestingly, the glycan present at N301 was earlier determined to be occupied by a tetraantennary complex glycan while most other complex type N-glycans are predominantly diantennary . This finding may raise the possibility that a multivalent interaction with more than two antennae is favourable for GNAmaize binding, although a glycan array revealed that GNAmaize showed the highest binding affinities to biantennary (or monoantennary) GlcNAc β1-2Man-containing glycans . In contrast, HIV-1 exposure to GNA resulted in the eventual deletion of 7 glycosylation sites of which 5 were high-mannose-type N-glycans (N230, N234, N289, N339 and N392) and only 2 complex-type N-glycans (N88 and N301) . Similar preference for the deletion of high-mannose-type glycans has also been observed for the Hippeastrum hybrid (Amaryllis) lectin HHA , the prokaryotic lectin actinohivin [37, 38], the cyanobacterial lectin Cyanovirin N , the 2G12 mAb  and the antibiotics pradimicin A and S [41, 42]. Such unusual preference for deletion of high-mannose-type glycans is highly significant for these lectins since the glycan shield of the HIV-1 gp120 envelope, determined for gp120 expressed in Chinese hamster ovary (CHO) cells, exists of 11 high-mannose- or hybrid-type glycans and 13 complex-type glycans . It was interesting to notice that one of the GNAmaize-exposed virus strains also showed a glycosylation site deletion in gp41. It should, however, be kept in mind that the N811 position is located in the cytoplasmic tail of gp41 and thus was not supposed to be glycosylated in wild-type gp41. The relevance of the appearance of this mutation is therefore unclear. Also, the relevance of the formation of the new glycosylation motif at N29 in gp120 of one of the virus isolates is unclear because this amino acid is located in the membrane-embedded signal peptide and thus unlikely to be used for glycosylation.
Fouquaert and colleagues  demonstrated by glycan array analysis that GNA strongly interacts with high-mannose-type N-glycans and preferentially recognizes terminal mannose residues (Manα1,6Man > Manα1,3Man > Manα1,2Man), whereas GNAmaize has poor, if any affinity for this type of glycans. In contrast, GNAmaize recognizes complex N-glycans with a preference for a GlcNAc β1,2Manα1,3-X motif-containing glycan and/or a Neu5Acα2,6Galβ1,4-X motif-containing glycan. Thus, this surprising shift in glycan specificity from high-mannose-type to complex-type glycans between the closely related GNA and GNAmaize explains the differences between both lectins in their preference for the nature (high mannose-type for GNA and complex-type for GNAmaize) of the deletion of N-glycans in the drug resistance selection experiments. To further document this shift in sugar recognition we performed several surface plasmon resonance (SPR) experiments. In the first instance 5 oligosaccharides: (α1,2-man)3, (α1,3-man)2, (β1,4-GlcNAc)3, GlcNAcß1,2Man and GlcNAcβ1,2Manα1,3(GlcNAcβ1,2Manα1,6) Manβ1,4GlcNAcβ1,4GlcNAc were examined for binding to immobilized GNA and GNAmaize. The SPR-results showed that only (α1,2-man)3 and (α1,3-man)2 preferentially bind to GNA but not GNAmaize whereas GlcNAcß1,2Man and GlcNAcβ1,2Manα1,3(GlcNAcβ1,2Manα1,6) Manβ1,4GlcNAcβ1,4GlcNAc were able to bind to GNAmaize but not to GNA. We found a slightly higher preference of GNA for (α1,2-man)3 than for (α1,3-man)2 whereas GNA was originally reported by Shibuya and co-workers  as a lectin with specificity towards oligosaccharides with terminal Manα1,3Man motifs. However, it should be noticed that in our SPR studies, a α1,3-man dimer but a α1,2-man trimer has been used. It is well known that often a higher degree of oligomerization results in a better affinity of the lectins for such sugar oligomers. The concomitant α1,2-man specificity of GNA is also in line with the glycan array data of Fouquaert et al. , and the α1,2-mannose oligomer affinity of GNA became also evident from the 2-fold lower KD-value of GNA binding to insect cell-derived gp120 (containing a high density of high-mannose-type glycan structures) than CHO cell-derived gp120 (Table 5). The 2-fold weaker affinity of GNAmaize against insect cell-derived gp120 compared to CHO-derived HIV-1 gp120 is also in line with its predominant complex-type glycan specificity.
Competition of GNA, GNAmaize and 2G12 mAb for binding to HIV-1 gp120
#RU at 2 min post injection
additional gp120 binding by the analyte (%)
5 μM GNA
409 ± 7
20 μM GNAmaize
111 ± 8
3 μM 2G12
313 ± 48
5 μM GNA + 20 μM GNAmaize
38 ± 4
34 ± 1.4
20 μM GNAmaize + 5 μM GNA
310 ± 6
76 ± 0.2
3 μM 2G12 + 5 μM GNA
287 ± 5
70 ± 0.0
5 μM GNA + 3 μM 2G12
78 ± 5
25 ± 5.4
3 μM 2G12 + 20 μM GNAmaize
93 ± 17
85 ± 21.3
20 μM GNAmaize + 3 μM 2G12
277 ± 4
89 ± 14.9
The Manα1,2-man oligomer-specific lectins [i.e. cyanovirin-N , Pradimicin A , Pradimicin S , actinohivin  and the mAb 2G12 ] and manα1,3/α1,6-man-oligomer specific lectins (i.e. GNA and HHA ) have previously been reported to contain potent anti-HIV activity. This manα1,2-, α1,3 or α1,6-man oligomer preference of GNA disappeared almost completely for the structurally closely related GNAmaize and, likewise, resulted in a seriously decreased antiviral activity and a markedly lower affinity for HIV-1 gp120. These findings reveal the importance of interaction of CBAs with high-mannose-type glycans (preferentially manα1,2man) on the HIV gp120 envelope protein as a prerequisite to exhibit pronounced antiviral activity. Although the designation of complex versus high-mannose-type glycans on gp120 is based on the study of Leonard et al.  using monomeric recombinantly expressed gp120, it is well possible that the glycan content of the native gp120 trimer on the viral particles is somewhat different. In fact, Doores et al.  recently revealed that the envelope of native HIV virions, in sharp contrast to recombinantly gp120, almost exclusively contains an oligomannose (Man5-9GlcNAc2) glycan profile (< 2% complex-type glycans). However, it should be kept in mind that a proportion of the high-mannose-type glycans determined on virion trimeric gp120 can be derived from non-functional envelope forms of the virus containing a different glycosylation profile and therefore the amount of high-mannose-type glycans on the gp120 of virus particles can somewhat be overestimated in this study.
In conclusion, the markedly reduced effect in anti-HIV activity (up to ~100-fold) of GNAmaize compared to GNA is explained by the shift in glycan recognition from high-mannose to complex-type glycans, and underscores the importance of efficient mannose-oligomer recognition of therapeutics as a prerequisite to exert significant anti-HIV activity. These findings would justify a rational design of new carbohydrate-binding therapeutics selectively targeting the high-mannose type glycans present on the HIV envelope gp120. Therefore, a better understanding of the molecular interaction between mannose-binding lectins such as actinohivin, cyanovirin, microvirin or griffithsin with α1,2-mannose oligomers by NMR or crystallography interaction studies would allow rational design of small synthetic carbohydrate (mannose)-binding agents. Also, (small-size) synthetic compounds such as borane-containing compound derivatives, known to specifically recognize configurations of two hydroxyl groups in cis (such as being present in mannose) [45, 46] should be explored for gp120 binding and anti-HIV activity.
This work was supported by the K.U. Leuven (GOA no. 10/014, Center of Excellence no. EF/05/15 and Program Financing no. PF/10/018), University of Ghent (BOF2007/GOA/0017) and the FWO (no. G.485.08). The authors are grateful to Leen Ingels, Becky Provinciael, Sandra Claes, Yoeri Schrooten, Lore Vinken and Romina Termote-Verhalle for excellent technical assistance, and Christiane Callebaut for dedicated editorial help.
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